Adjudin targeting rabbit germ cell adhesion as a male contraceptive: a pharmacokinetics study

Abstract

Adjudin (1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide; formerly called AF-2364) has been shown to inhibit spermatogenesis by disrupting anchoring junctions at the Sertoligerm cell interface. This, in turn, leads to germ cell loss from the seminiferous epithelium, and transient infertility. Adjudin's efficacyin inhibiting spermatogenesis, the recovery of spermatogenesis after cessation of the drug, and side effects were examined in adult male Japanese rabbits. The pharmacokinetics profiles of adjudin in rabbits after oral administration and after intravenous injection were compared. Rabbits received 25 mg/kg adjudin once weekly for 4 consecutive weeks either by intravenous injection or by gavage. Vehicle-treated rabbits were used as controls. At 1, 2, 3, 4, and 8 weeks after treatment, testes were removed for microscopic examination to assess the status of spermatogenesis. Four weeks after intravenous cessation of adjudin, the recovery of spermatogenesis also was monitored. Blood was withdrawn after first administration to measure plasma concentrations of adjudin by high-performance liquid chromatography. Four weeks after intravenous treatment, examination of testis sections showed rapid exfoliation of elongated/elongating spermatids and the presence of large multinucleated cells; more than 95% of germ cells were absent from the seminiferous epithelium. Intravenous treatment showed a more severe disturbance of spermatogenesis compared with gavage treatment, which was correlated with bioavailability of the drug. The areas under the curve for intravenous injection and gavage were 20.11 +/- 1.90 and 2.23 +/- 0.45 mg x h x L(-1), respectively. These results illustrate the potential of adjudin as a male contraceptive, and the efficacy is associated with the bioavailability of the drug.

Figure 1

Chromatographic separation of adjudin and carbamazepine (internal control). Adjudin and carbamazepine peaks were well separated. (A) Chromatograph of carbamazepine (peak 1, retention time 4.7 minutes) and adjudin (peak 2, retention time 7.2 minutes) dissolved in mobile phase (x-axis is the retention time in minutes and y-axis is the value of absorbance at 302 nm). (B) Chromatograph of blank plasma without adjudin and/or carbamazepine. (C) Chromatograph of adjudin and carbamazepine in a rabbit plasma sample. mAU indicates milli-absorbance unit.

Figure 2

Standard curve of adjudin. This is estimated by the highperformance liquid chromatography method as described in “Materials and Methods.” Ai indicates absorbance of adjudin; As, absorbance of the internal standard, carbamazepine.

Figure 3

Plasma time curve of adjudin after intravenous or oral administration of single dose of 25 mg/kg adjudin on 6 male Japanese rabbits. (A) Adjudin plasma profile after intravenous administration. (B) Adjudin plasma profile after gavage. Adjudin level in plasma was estimated by HPLC as shown in Figures 1 and 2.

Figure 4

Testis sections after intravenous or oral administration of adjudin. Adult rabbits were treated with adjudin (25 mg/kg b.w.) intravenously or orally and killed 2, 3, 4, or 8 weeks after treatment for histologic analysis by hematoxylin and eosin staining. Cross section of control (A), 2 (B) or 4 (C) weeks after IV administration, 4 weeks (D) after oral administration, or 4 weeks after cessation of adjudin by intravenous (E) or oral (F) administration. Scale bar 5 50 mm.

Figure 5

Epididymis sections after intravenous or oral administration of adjudin. Adult rabbits were treated with adjudin (25 mg/kg b.w.) intravenously or orally and killed 2, 3, 4, or 8 weeks after treatment for histologic analysis by hematoxylin and eosin staining. Cross section of control (A), 2 (B) or 4 (C) weeks after intravenous administration, 4 weeks (D) after oral administration, or 4 weeks after cessation of adjudin by intravenous (E) or oral (F) administration. Scale bar = 50 μm.